Modified beta-lactamase and method for its preparation

ABSTRACT

The invention relates to targeted post translational modifi-cation of metallo-beta-lactamase by truncation and inser-tion of a dipeptide at the amino terminal end to reduce amino terminal heterogeneity in a recombinant DNA pro-duction system. A protein K-T-E-ΔBL is expressed, and modified by host proteases to E-ΔBL. Appropriate nucleotide molecules, vectors and hosts are also de-scribed. E-ΔBL is useful in a pharmaceutical composition for treating antibiotic induced adverse effects in the intes-tine of patients treated with beta-lactam antibiotics.

FIELD OF THE INVENTION

Various antibiotics are used in the treatment of bacterial infections.However, the antibiotics do not only attack pathogens, but they alsoaffect the normal bacterial flora, leading to adverse side effects e.g.in the patient's intestine. These side effects can be reduced byadministering enzymes capable of degrading residual antibiotic in theintestine. The present invention relates to modifiedmetallo-beta-lactamases that are useful in treating and preventingadverse effects of antibiotics having a beta-lactam ring, or in thepreparation of such enzymes. The invention is also directed to a methodfor preparing the modified beta-lactamases as well as to nucleotidemolecules, vectors and host cells useful therein.

BACKGROUND OF THE INVENTION

Beta-lactamase enzymes represent a major mechanism of resistance amongbacteria to beta-lactam antibiotics, which include penicillins,cephalosporins and carbapenems. These enzymes catalyse the irreversiblehydrolysis of the amide bond of the beta-lactam ring to createineffective antimicrobial agents. On the basis of molecular structureclassification and catalytic mechanisms the beta-lactamases can bedivided into four classes: A, B, C, and D. Classes A, C and D are serineenzymes and comprise the majority of the beta-lactamases (Ambler, 1980).These enzymes generally inactivate penicillins or cephalosporins andoften show a preference for one of these two antibiotics.

Class B beta-lactamases are metallo-enzymes that require one or two zincions as a cofactor for enzyme activity. The metallo-beta-lactamasesconstitute group 3 in the Bush-Jacoby-Madeiros functional classification(Bush, 1998). This schema is primarily based on substrate profiles,their sensitivity to EDTA and their resistance to serine beta-lactamaseinhibitors. Based on structural similarities in the region thatcoordinates zinc binding, metallo-beta-lactamases can be divided intothree subgroups, B1, B2, and B3 (Galleni et al., 2001). Subgroup B1possesses three histidines and one cysteine as the key zinc coordinatingresidues. Crystallographic structures have been described for manysubgroup B1 enzymes such as BcII of Bacillus cereus (Carfi et al, 1995and 1998a), CcrA of Bacteroides fragilis and (Carfi et al., 1998b) andIMP-1 of Pseudomonas aeruginosa (Concha et al., 2000). Correspondingly,subgroup B2 lactamases have an arginine residue, instead of histidine,at the first position of the principal zinc binding motif, NXHXD.Recently, the first crystal structure of a subgroup B2 enzyme (CphA) hasbeen solved by Garau et al. (2005). Subgroup B3 contains enzymes withmultimeric structure (Walsh et al., 2005).

Metallo-beta-lactamases show a broad spectrum substrate profileincluding penicillins and cephalosporins, and they are resistant to theaction of common conventional serine beta-lactamase inhibitors such asclavulanic acid sulbactam and tazobactam. Furthermore, unlike most ofthe serine beta-lactamases, metallo-beta-lactamases have the capabilityto hydrolyze carbapenems such as meropenem and imipenem. Various numbersof bacteria are known to produce metallo-beta-lactamases. They arecommonly expressed among the Enterobacteriae genus (including Serratiamarcescens, Klebsiella pneumoniae, Citrobacter freudii, Shigellaflexneri), Pseudomonas aeruginosa, Stenobacterium maltophila,Acinetobacter genus, Bacteroides fragilis, Bacillus cereus,Flavobacteruim odoratum, and Bacteroides fragilis (Walsh et al., 2005).

Beta-lactamases can be utilized as pharmaceutical proteins to inactivateunabsorbed beta-lactams in the gastro intestinal tract in order toprevent the beta-lactam induced adverse effects including alterations inintestinal normal microbiota and the overgrowth of beta-lactam resistantbacteria (WO93/13795, WO2004/016248). For efficient beta-lactamasetherapy in the small intestinal tract the enzyme should be resistant tothe action of intestinal proteases in the presence of bile acids andpreserve high enzymatic activity at a wide range of pH (5.5-7.5).

The feasibility of targeted enzyme therapy in canine and mouse modelswas demonstrated by employing a Bacillus licheniformis serinebeta-lactamase during parenteral ampicillin medication (Harmoinen etal., 2004, Mentula et al., 2004, Stiefel et al., 2003). However, thesubstrate profile of this enzyme essentially limits its use as a drugsubstance since it has poor capacity to hydrolyze cephalosporins,carbapenems or penicillins in the presence of beta-lactamase inhibitors.Consequently, a new protease resistant beta-lactamase enzyme with broadbeta-lactam spectrum is indispensable to extend the use ofbeta-lactamase therapy among hospitalized patients under intravenousmedication with various beta-lactams.

Metallo-beta-lactamases are known to inactivate various types ofbeta-lactams and they are resistant to inhibitors of serinebeta-lactamases. Bacillus cereus strains are known to producemetallo-beta-lactamase that belongs to group B1. A semi purifiedrecombinantly produced metallo-beta-lactamase sample of a clinicalBacillus cereus 98ME1552 isolate was shown to eliminate the overgrowthof potential pathogenic bacteria in a mouse model (Stiefel et al.,2005). However, the present inventors found that thismetallo-beta-lactamase preparation contained a mixture of beta-lactamasevariants, which declines its value as a pharmaceutical protein, sincevariations of a drug substance reduce the robustness of the productionprocess, increase batch to batch variations, and make clinical trialsdifficult, which of course has a negative impact on its registration asa medicament.

The present invention now provides means for reducing the amino terminalheterogenicity that was found to be associated with recombinantproduction of metallo-beta-lactamase. The invention further providesmodified metallo-beta-lactamases that can be produced in substantiallypure form and that can be used in the manufacture of pharmaceuticalcompositions.

SUMMARY OF THE INVENTION

The invention provides a modified metallo-beta-lactamase protein havingthe general formula:

NH₂—K-T-E-ΔBL-COOH  (I)

wherein

K is lysine

T is threonine

E is glutamic acid, and

ΔBL is a metallo-beta-lactamase protein that has been truncated at theamino terminal end so as to leave four beta-strands before the firstalpha-helix of the predicted secondary structure of said protein.

The invention further provides an isolated nucleotide moleculecomprising a nucleotide sequence encoding said modifiedmetallo-beta-lactamase protein, as well as an expression vectorcontaining the nucleotide molecule, and a host cell capable ofexpressing the metallo-beta-lactamase protein encoded by the nucleotidemolecule.

The invention also provides a modified metallo-beta-lactamase proteinhaving the general formula

NH₂-E-ΔBL-COOH  (II)

wherein

E and ΔBL are as defined above.

The invention still further provides a method of preparing a modifiedmetallo-beta-lactamase protein, said method comprising culturing saidhost cell under conditions enabling the expression of ametallo-beta-lactamase having the general formula:

NH₂—K-T-E-ΔBL-COOH,  (I)

wherein

K is lysine

T is threonine

E is glutamic acid, and

ΔBL is a metallo-beta-lactamase protein that has been truncated at theamino terminal end so as to leave four beta-strands before the firstalpha-helix of the predicted secondary structure of said protein,

and conducting post translational modification resulting in a modifiedmetallo-beta-lactamase having the general formula:

NH₂-E-ΔBL-COOH,  (II)

wherein E and ΔBL are as defined above, and

optionally isolating and purifying the post translationally modifiedprotein obtained.

As additional aspects the invention provides a pharmaceuticalcomposition comprising the modified metallo-beta-lactamase protein offormula II, the modified metallo-beta-lactamase of formula II for use asa medicament, and the use of the modified metallo-beta-lactamase offormula II for the manufacture of a medicament for elimination ofbeta-lactam antibiotic induced adverse effects in the intestinal tract.

Finally the invention provides a method of treating beta-lactamanti-biotic induced adverse effects in the intestinal tract comprisingadministering an effective amount of the modified metallo-beta-lactamaseof formula II, or the pharmaceutical composition containing it, to aperson in need thereof. Specific embodiments of the invention are setforth in the dependent claims.

Other objects, details and advantages of the present invention willbecome apparent from the following drawings, detailed description andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the complete nucleotide sequence and the deduced amino acidsequence of the B. cereus 98ME1552 beta-lactamase gene.

FIG. 2 shows the complete nucleotide sequence and the deduced amino acidsequence of the B. cereus 98ME1552 beta-lactamase gene derived from thepRSH315 expression construct. The cleavage site of the 31 amino acidresidues long signal sequence of Bacillus amyloliquefaciens is predictedto occur between alanine at position −1 and glutamine at position +1.The NH₂-terminal extension of a NH₂-QAS-tripeptide that is derived fromthe Hind III cloning site is expressed in bold.

FIG. 3 shows the complete nucleotide sequence and the deduced amino acidsequence of the B. cereus 98ME1552 beta-lactamase gene derived from thepRSH318 expression construct. The cleavage of a 31 amino acid residueslong signal sequence of Bacillus amyloliquefaciens is predicted to occurbetween alanine at position −1 and glutamine at position +1. TheNH₂-terminal extension of the NH₂-QAS-tripeptide derived from the HindIII cloning site and the KT insertion are shown in bold.

DETAILED DESCRIPTION OF THE INVENTION

A metallo-beta-lactamase preparate was first produced in a Bacillussubtilis production system containing an expression construct of thecomplete metallo-beta-lactamase gene encoding the complete metallo-betaenzyme. Detailed mass spectrometry analysis revealed that the metalloenzyme preparate included various numbers of enzyme variants withheterogeneous amino terminal sequences. The variations at the aminoterminus sequences were believed to be a result of post translationalmodification of host cell proteases. The observed alterations in theamino acid sequence of the metallo enzyme increase batch to batchvariations of enzyme that has been produced in a Bacillus subtilisproduction system and thus in regulatory perspective reduce its use as apharmaceutical protein. Therefore molecular biological means forreducing the observed variations in the amino terminal sequence of themetallo-beta-lactamase enzyme were studied.

The amino terminal region is expected to have no essential influence onthe catalytic properties of the enzyme. In addition the amino terminalregion was found to be exposed to post translational modifications ofproteases in the Bacillus subtilis production system in which therecombinant protein is secreted outside the bacterial cell. In order toreduce this microheterogeneity in the amino terminal region, thenucleotide sequence encoding this predicted region was deleted by a PCRmethod. However, mere deletion by itself did not lead to a significantreduction of the amino terminal heterogeneity. Surprisingly, however,the deletion combined with insertion of a dipeptide, which was designedto assist post translational modification, led to a singlemetallo-beta-lactamase variant produced with an equally modified aminoterminus.

This invention relates in general to a modified beta-lactamase useful asa pharmaceutical protein, and also to a recombinant beta-lactamaseintermediate that is produced by truncation and insertion of a dipeptideat its amino terminal resulting in reduced numbers of beta-lactamasevariants. These modifications facilitate the recombinant production ofthe enzyme in homologous form for use as a drug substance inbeta-lactamase therapy for elimination of beta-lactams (cephalosporins,carbapenems, and penicillins in the presence or absence of knownbeta-lactamase inhibitors such as clavulanic acid, sulbactam andtazobactam) induced side effects. In particular the invention relates totargeted post translational modification of activemetallo-beta-lactamase by truncation and insertion of a dipeptide at theamino terminal region to reduce amino terminal heterogeneity in aBacillus subtilis production system.

“Metallo-beta-lactamases” as used herein refer to class Bbeta-lactamases i.e. beta-lactamases that require at least one bivalentmetal ion (Zn²⁺) for activity; and constitute group 3 in the in theBush-Jacoby-Madeiros functional classification (Bush, 1998). Based onthe structural similarities in the region that coordinates zinc binding,metallo-beta-lactamases can be divided into three subgroups, B1, B2, andB3 (Galleni et al., 2001). Preferably the metallo-beta-lactamase of theinvention belongs to subgroup B1. This subgroup possesses the key zinccoordinating residues of three histidines and one cysteine. More detailsabout the subgroups are set forth in the background part of thisspecification.

The metallo-beta-lactamases are members of a large, diverse, superfamilyof proteins that share a similar four layered αβ/βα structure. Thepolypeptide chain is divided into two domains, which comprise strandsand helices in the following order: β₁ β₂ β₃ β₄ β₅ α₁ β₆ α₂ β₇ α₃ and β₈β₉ β₁₀ β₁₁ α₄ β₁₂ α₅, respectively (Carfi et al., 1995 and 1998a, andGalleni et al., 2001). The metallo-beta-lactamases of the invention aretruncated at the amino terminal end prior to the second beta strand.Preferably they are truncated between the first and second beta strands,and in particular immediately in front of the amino acid E between thefirst and second beta strands according to the crystallographicstructure above.

Garau et al., 2004 have set forth a standard numbering scheme for classB beta-lactamases (BBL) by analogy to Ambler beta-lactamase numbering(ABL) for serine beta-lactamases (Ambler, 1980). The BBL numberingscheme is derived from a structural alignment of knownmetallo-beta-lactamase X-ray structures in order to discover conservedregions among various metallo beta-lactamase groups having low degree ofidentity in primary amino acid sequence level. The study of Garau et al.revealed that the first conserved fragment forms the second beta sheetstructure in Bacillus cereus BcII metallo beta-lactamase set forth byCarfi et al. 1995, and Carfi et al., 1998a. Consequently, the β₁-sheetof B. cereus metallo beta-lactamase appears to be non-essential forenzyme function, because it is not be present in all metallobeta-lactamase groups. However, the amino terminal region of all metallobeta-lactamases possess a secondary structure of four conserved betasheets forming fragments before the first alpha helix forming fragment.Thus the truncation site for the metallo beta-lactamase may be definedas one leaving four beta strands before the first alpha helix of thepredicted secondary structure regardless of the original presence of β₁or not.

In the present context conventional one-letter amino acid codes and areused. Thus, A denotes alanine, R denotes arginine, N denotes asparagine,D denotes aspartic acid, C denotes cysteine, E denotes glutamic acid, Qdenotes glutamine, G denotes glycine, H denotes histidine, I denotesisoleucine, L denotes leucine, K denotes lysine, M denotes methionine, Fdenotes phenylalanine, P denotes proline, S denotes serine, T denotesthreonine, W denotes tryptophan, Y denotes tyrosine, and V denotesvaline. The amino acid sequences are shown with the amino terminus tothe left and carboxyl terminus to the right. NH₂— and —COOH may or maynot be indicated.

According to one embodiment of the invention a host cell is transformedwith an expression vector capable of expressing a beta-lactamase havingthe general formula I:

NH₂—K-T-E-ΔBL-COOH,  (I)

wherein K is lysine, T is threonine, E is glutamic acid, and ΔBL is ametallo-beta-lactamase protein that has been truncated at the aminoterminal end prior to the second beta-strand of said protein, whereby aprotein having the general formula II:

NH₂-E-ΔBL-COOH,  (II)

is formed as a result of post translational modification of the proteinof formula I. The protein of the general formula I is convenientlyproduced by ligating a KT encoding DNA sequence to an E-ΔBL encoding DNAsequence, wherein E-ΔBL is a Bacillus and in particular a B. cereusmetallo-beta-lactamase that has been truncated at the amino terminal endprior to the second beta strand immediately in front of a glutamic acidresidue E. In particular the nucleotide construct comprises thesequential nucleotides 94-756 of SEQ ID NO: 2.

The length of the amino terminal region without rigid structure variesbetween metallo-beta-lactamase groups and within each subclass. Themetallo-beta-lactamases of Bacillus spp also possess variations in theamino terminal sequence prior to the second beta strand, but mostenzymes have a conserved glutamic acid (E) at position +12 (see Table1). Consequently, the deletion coupled with the KT insertion adjacent toE can be applied to known Bacillus metallo-beta-lactamases, in whichcase the first eleven amino terminal amino acids are deleted from theN-terminal end of the naturally occurring mature beta-lactamase protein.According to one specific embodiment of the invention the beta-lactamaseis truncated between the amino acids KT and E, and especially betweenVIKN and E, or VMKN and E.

TABLE 1 Comparison of the amino terminal sequences between Bacillus spmetallo-beta-lactamases and P2A.

*The amino acid substitutions are bolded and the conserved glutamic acid(E) is shaded. The first beta strand for metallo-beta-lactamases is onceunderlined, and the second beta strand is twice underlined.

According to a specific embodiment of the invention E-ΔBL has at least70, 80, 90, 95, 98 or 99% identity to the amino acid sequence ofsequential amino acid residues 6-221 of SEQ ID NO: 3. Sequence identitymay be determined using BLAST (Basic Local Alignment Search Tools) asdescribed in Altschul et al., 1997. In particular E-ΔBL is a truncatedform of a metallo-beta-lactamase having the sequence as set forth as SEQID NO: 1, or a beta-lactamase active variant or fragment thereof. It maye.g. be a truncated form of B. cereus metallo-beta-lactamase BcII.Preferably E-ΔBL has an amino terminal end that corresponds to aminoacid residues 12-15, 12-19, or 12-23 of SEQ ID NO: 1, with the additionthat the amino acid at position +13 may be either T (threonine) or A(alanine).

By an amino acid sequence that is a “variant” of a specific amino acidsequence is meant an amino acid sequence that is not identical to thespecific amino acid sequence, but contains at least some amino acidchanges i.e. deletions, substitutions, inversions, insertions etc. thatdo not essentially affect the biological activity of the protein ascompared to that of the specific amino acid sequence. Biologicalactivity in this context refers to beta-lactamase activity. A variantmay be a polypeptide that occurs naturally e.g. as an allelic variantwithin the same strain, species or genus, or it may have been generatedby mutagenesis.

A “fragment” is understood to be part of a specific amino acid sequencethat is long enough to have the desired biological activity i.e.beta-lactamase activity. In other words the fragment may be e.g. asubsequence of the specifically disclosed beta-lactamases.

The nucleotide construct encoding NH₂—K-T-E-ΔBL-COOH may be inserted inan expression vector, and transformed into a host cell. The host cell isthen cultivated under conditions enabling expression and posttranslational modification of the protein into NH₂-E-ΔBL-COOH, whichthus may be obtained in substantially pure form, which means that atleast 90%, and preferably at least 95%, in particular at least 99% ofthe metallo-beta-lactamase is present in a single form asNH₂-E-ΔBL-COOH. The post translational modification of the expressedprotein, i.e. the cleavage of the dipeptide KT is catalyzed by proteasesof the host. The host may be eukaryotic or prokaryotic, such as abacterial, yeast or fungus cell. The bacterial host may be e.gEscherichia coli. Preferably the host belongs to Bacillus spp, and inparticular it is B. licheniformis or B. subtilis. According to onepreferred embodiment the NH₂—K-T-E-ΔBL-COOH is expressed as a proteincomprising a signal peptide, whereby the protein is secreted into theculture medium, and the signal peptide is first cleaved, and then thedipeptide. Minor other changes such as deamidation, oxidation, disulfidebond breakage or formation, isomerization, succinimidation,non-disulfide crosslinking, Maillard reaction, deglycosylation may occurin the protein during the production process or storage, and areacceptable as long as the beta-lactamase activity is not significantlyaffected.

The modified beta-lactamase NH₂-E-ΔBL-COOH may originate from anybacterium capable of producing a metallo-beta-lactamase. Such bacteriaare e.g. bacteria of the Enterobacteriae genus (Serratia marcescens,Klebsiella pneumoniae, Citrobacter freudii, Shigella flexneri),Pseudomonas aeruginosa, Stenobacterium maltophila, Acinetobacter genus,Bacteroides fragilis, Bacillus cereus, Flavobacteruim odoratum, andBacteroides fragilis. Other possible sources are Aeromonas, Legionellaand Stenotrophomonas species. The enzyme is truncated at the aminoterminal end of the mature enzyme protein prior to the second betastrand at a position resulting in E as the N-terminal amino acid. Ifthere is no appropriate residue E in the bacterial beta-lactamase, onlythe ΔBL part may be obtained from the bacterium, whereas a tripeptideKTE is coupled in front of ΔBL to from the protein NH₂—K-T-E-ΔBL-COOH.

Preferably the modified beta-lactamase is derived from Bacillus, andespecially from B. cereus. In particular it is a B. cereusbeta-lactamase from which the first eleven N-terminal amino acids of thenon-modified mature beta-lactamase protein have been deleted.

The modified beta-lactamase of the invention may be mixed with anypharmaceutically acceptable excipients or carriers to form apharmaceutical composition. An amount of the modified beta-lactamaseeffective of eliminating the adverse effect of beta-lactam antibioticsin the intestine is then administered orally to a person being treatedwith one or more beta-lactam antibiotics. The beta-lactamase preparationmay be administered before, simultaneously with, or after the antibiotictreatment. It is insensitive to serine beta-lactamase inhibitors, andmay therefore be used to eliminate the antibiotic both in the presenceand absence of such inhibitors.

The invention is illustrated by the following non-limiting examples. Itshould be understood, however, that the embodiments given in thedescription above and in the examples are for illustrative purposesonly, and that various changes and modifications are possible within thescope of the invention.

Example 1 Materials and Methods Bacterial Strains and Growth Conditions

The bacterial strains and their relevant genotypes and phenotypes arepresented in Table 2.

TABLE 2 Bacterial strains and their relevant genotypes and phenotypesStrain Relevant genotype Relevant phenotype Bacillus subtilis RS303trpC2, sigG::cat Tryptophan auxotroph, (Δ sigG) asporogenic Bacillussubtilis IH 6140 sacA321 Reduced exoprotease level Bacillus subtilisRS314 trpC2, sigG::cat, (Δ sigG) Tryptophan auxotroph, pRSH314expression construct asporogenic, secretes encoding the truncatedmetallo-beta-lactamase metallo-beta-lactamase Bacillus subtilis RS315trpC2, sigG::cat, (Δ sigG) Tryptophan auxotroph, pRSH315 expressionconstruct asporogenic, secretes encoding the completemetallo-beta-lactamase metallo-beta-lactamase gene Bacillus subtilisRS317 trpC2, sigG::cat, (Δ sigG) Tryptophan auxotroph, pRSH317expression construct asporogenic, secretes encoding truncatedmetallo-beta-lactamase metallo-beta-lactamase Bacillus subtilis RS318trpC2, sigG::cat, (Δ sigG) Tryptophan auxotroph, pRSH318 expressionconstruct asporogenic, secretes encoding truncatedmetallo-beta-lactamase metallo-beta-lactamase Bacillus cereus 98ME1552Multiresistant to various beta-lactams

Bacteria were generally cultivated in complex Luria medium (5 grams ofyeast extract, 10 grams of tryptone and 10 grams of sodium chloride perlitre) supplemented with appropriate antibiotics as selection agents inshaking flask mode at +37° C. A synthetic culture medium supplementedwith appropriate antibiotic was used in fermentations. A modifiedsynthetic medium was used to generate competent Bacillus subtilis cells.Detailed composition of these media is described in WO03/040352.

DNA Techniques

Conventional DNA techniques including e.g. restrictions, agarose gelelectrophoresis and ligations, were performed according to Sambrook andRussell (2001). The chromosomal DNA was isolated by the Marmur method(Marmur, 1961). The plasmid DNA was isolated by Qiagen plasmid Midi Kitaccording to manufacturer's instructions (Qiagen Plasmid PurificationHandbook, 1999) but lysozyme treatment (1 mg/ml) was added to theprotocol in order to degrade the peptidoglycan layer. Lysozyme wassupplemented to buffer P1 and cells were incubated at 37° C. for 30minutes.

PCR was generally performed according to protocols described inWO03/040352.

Characterization of Amino Terminal Region of Various Forms ofMetallo-Beta-Lactamase

For determinations of the N-terminal amino acid sequence and massspectrometry, purified metallo-beta-lactamase samples were subjected toreverse phase chromatography and the enzymes were fractioned in a lineargradient of 0.1% of TFA and 0.075% TFA-acetonitrile from 0% to 100% for60 minutes.

NH₂-terminal sequences of metallo-beta-lactamase forms were determinedby automated Edman degradation with an Applied Biosystems model 494AProtein Sequenator.

Mass analyses of metallo-beta-lactamase forms were performed by a hybridquadrupole-time-of-flight (Q-TOF) instrument. Metallo-beta-lactamasevariants have been assumed to provide comparable ionization potentialswhich have been utilized in the assessments of their relativeproportions in the samples.

The nucleotide sequences of the beta-lactamase genes were determined bythe dideoxy-chain termination method with an automatic DNA sequencer.

Example 2 The Complete Nucleotide Sequence of Bacillus cereus 98ME1552Metallo-Beta-Lactamase Gene

The determination of the complete gene coding metallo-beta-lactamase wassequentially performed by using PCR and Vectorette techniques. Part ofthe structural gene was amplified by PCR, with isolated chromosomal DNAof a clinical isolate B. cereus 98ME1552 as a template and with primersdesigned to hybridize to the SQKVEKTVI coding region (forward primer)and HTLDLL coding region (reverse primer) of the Bacillus cereus 569/Hmetallo-beta-lactamase gene. Both primers also carry Hind IIIrestriction sites. The amplified DNA fragment (about 700 bp) wasdigested with Hind III and ligated to the Hind III site of a secretionvector pKTH141. Competent Bacillus subtilis IH6140 cells weretransformed with the ligation mixture. A clone harbouring a plasmidexpressing metallo-beta-lactamase was verified by DNA sequencing. Theplasmid was named pRSH314.

Competent B. subtilis RS303 cells prepared as described in WO03/04052were transformed with pRSH314 resulting in strain B. subtilis RS314.

The complete DNA sequence of metallo-beta-lactamase gene was determinedfrom PCR fragments obtained by employing Vectorette technique asfollows: The chromosomal DNA of Bacillus cereus 98ME1552 was digestedwith Hind III restriction enzyme and ligated to Hind III treatedVectorettell. The obtained Vectorette-library was screened in a PCRreaction by employing the MEBLSQ-F (5′-AGGAAATGTTGCGGATGC) or MEBLSQ-R(5′-CCTTCGTTAATTTGTTATCCC) initiating primers designed from the DNAsequence obtained from the 700 bp of pRSH314.

PCR screening of the Vectorette library with the MEBLSQ-F primergenerated a fragment of about 1000 bp (MEBL1 fragment) and with theMEBLSQ-R primer a fragment of about 1100 bp (MEBL2 fragment). Both MEBL1and MEBL2 fragments were purified from agarose gel afterelectrophoresis. The nucleotide sequences of both fragments weredetermined by DNA sequencing resulting in the complete nucleotide of B.cereus 98ME1552 metallo-beta-lactamase gene. The complete nucleotide anddeduced amino acid sequence of the B. cereus 98ME1552metallo-beta-lactamase gene is presented in FIG. 1. The amino acidsequence is also set forth as SEQ ID NO: 1. The open reading frameencodes a 257 amino acid polypeptide, whose amino terminal sequence (30amino acid residues) exhibits features typical of those of a bacterialsignal peptide that targets protein secretion across the cytoplasmicmembrane via a general secretory pathway. The predicted signal peptidasecleavage site is after alanine at position of −30 (see FIG. 1). Thecalculated molecular mass and the pI value of the mature protein of 227amino acid residues is 24 877.3 Da and 6.0, respectively. The 98ME1552metallo-beta-lactamase sequence Was used as a search template in a BLASTsearch in order to identify the protein. The BLAST search was performedusing the SIB BLAST Network Service of Swiss Institute of Bioinformatics(http://www.expasy.org/tools/blast/). The search was done against aUniProtKB Knowledge based database using the NCBI BLASTP 2.2.13 program(Altschul et al., 1997) with BLOSUM62 algorithm and gap penalties 11 forexistence and 1 for extension. The BLAST search performed with the98ME1552 metallo-beta-lactamase gave the highest similarity scored withother class B beta-lactamases of subgroup B1. As expected, the 98ME1552beta-lactamase exhibited a high degree of identity (over 90%) to otherB. cereus beta-lactamases. According to structural alignment of variousB. cereus metallo-beta-lactamase, the amino acid substitutions in B.cereus 98ME1552 lactamase are predicted to locate in regions notessential for the function of the enzyme.

Example 3 Cloning and Expression of the B. cereus 98ME1552Metallo-Beta-Lactamase Gene in Bacillus subtilis

Based on the obtained DNA sequences from the Vectorette library, newprimers,

BLC1-F (5′-CGCGAAGCTTCCGAACAAAAGCTAGAGCAAATAGTAATC), and BLC1-R (5′-GCCGAAGCTTTTATTTTAATAAATCCAATGTATGTAAAAGTAATCCC)were designed to generate a DNA insert encoding the completemetallo-beta-lactamase in PCR. The primers also carried Hind III-sitesat their ends and purified chromosomal DNA of B. cereus 98ME1552 wasused as a template. The amplified PCR fragment of about 0.7 kb wasdigested with Hind III and ligated to the Hind III site of a pKTH141secretion vector. Competent Bacillus subtilis RS303 cells weretransformed by the ligation mixture. One positive clone was designatedRS315 and the harbouring expression construct was named pRSH315.

The pRSH315 expression construct was isolated and the insert region wassequenced. The nucleotide and deduced amino acid sequences of the clonedmetallo-beta-lactamase gene were identical to those determined from theVectorette library. The determined DNA sequence revealed in frame fusionbetween the nucleotide sequence encoding a 31 amino acid long signalsequence of Bacillus amyloliquefaciens alpha amylase, the Hind IIIcloning site and the complete B. cereus 98ME1552 metallo-beta-lactamasegene (see FIG. 2). Signal peptidase is predicted to cut the peptide bondbetween alanine (A) at position of −1 and glutamine (Q) at position of+1. The mature metallo-beta-lactamase possesses an NH₂-terminalextension of a NH₂-QAS-tripeptide that is derived from the Hind IIIcloning site in the expression construct. Hence, based on the deducedamino acid sequence the mature modified metallo-beta-lactamase iscomprised of 230 amino acid residues.

Example 4 Determination of the Amino Terminal Amino Acid Sequence ofMetallo-Beta-Lactamase Variants Obtained from a Bacillus subtilisProduction System

For further characterization, the recombinant metallo-beta-lactamase wasproduced in cultivations performed in either shaking flask orfermentation by using a synthetic growth medium. Themetallo-beta-lactamase was effectively secreted into the culturesupernatant. The enzyme was purified from concentrated culturesupernatant by ion exchange chromatography. Enzyme activity was observedin two separate fractions. Purified enzyme fractions were subjected toNH₂-terminal sequencing and mass spectrometry analysis. Analysesrevealed that both fractions comprised enzyme forms with heterogeneousamino terminus. The various enzyme forms and their relative proportionsare presented in Table 3. Related to the deduced amino acid sequence,all enzyme forms have deletions of various lengths in their NH₂-terminalregions. In general, the NH₂-QASEQKLE octapeptide seems to be deleted inall enzyme forms. Furthermore the smallest enzyme form in fraction 2lacks an additional IVIKN-pentapeptide. The observed microheterogeneityin the NH₂-terminal region can be explained as post translationalmodifications caused by the action of various host cell proteases.

TABLE 3 Deduced and determined amino terminal sequences and determinedmolecular mass of truncated metallo-beta-lactamase forms DeterminedRelative Enzyme NH₂- proportion of fraction Deduced NH₂-terminalterminal enzyme forms Determined n:o amino acid sequence sequence infractions (%) Mass (kDa) 1 NH₂-QASEQKLEQIVIKNETGTI NH₂-IVIKNETGTI 10024.122 2 NH₂-NETGTI 60 23.668 2 NH₂-ETGTI 40 23.554

Example 5 Construction of Bacillus cereus 98ME1552 DeletedMetallo-Beta-Lactamase Form and its Amino Terminal Variants in aBacillus subtilis Production System

In order to try to reduce NH₂-terminal heterogeneity the DNA sequenceencoding the variable region NH₂-EQKLEQIVIKN of themetallo-beta-lactamase gene was deleted by PCR. The complete gene of B.cereus 98ME1552 metallo-beta-lactamase was used as a template in PCRwith a forward primer hybridizing to a ETGTISISQ coding sequence and areverse primer hybridizing to a GLLLHTLDLLK coding sequence followed bya translation stop codon TAA. Both primers carried Hind III sites.

The obtained PCR fragment of about 0.7 kb was cloned into the Hind IIIsite of the pKTH141 secretion vector and the competent B. subtilis RS303strain was transformed by the ligation mixture. The NH₂-terminaldeletion was verified by DNA sequencing of the insertedmetallo-beta-lactamase gene of the expression construct isolated from apositive clone. The transformant strain was named Bacillus subtilisRS317 and the expression construct was called pRSH317.

Truncated metallo-beta-lactamase was produced in a Bacillus subtilisproduction host and the enzyme was purified from the culture supernatantby employing ion change chromatography from which the active enzyme waseluted as a single fraction. The enzyme fraction was subjected toNH₂-terminal amino acid sequencing and molecular mass analysis. Theobserved results (in Table 4) showed that the truncatedmetallo-beta-lactamase also appeared as a protein with a heterogeneousamino terminus. The major enzyme variant possesses an amino-terminalsequence identical to that of the deduced amino acid sequence includingthe Hind III site coding the initiation QAS-tripeptide. A minor variantwas found to have a deletion of a QA-dipeptide. Due to amino terminalblockage, no amino acid sequence was received from one variant. Aminoterminal blockage is likely to be the result of cyclization of theglutamine residue to form a pyroglutamyl residue during the productionprocess. To conclude, the amino terminal deletion did not fundamentallyreduce amino terminal microheterogeneity.

TABLE 4 Determined amino terminal sequences and molecular masses oftruncated metallo-beta-lactamase forms. Deduced NH₂- Relative pro-Enzyme terminal portion of fraction amino acid Determined NH₂- enzymeforms Mass analysis n:o sequence terminal sequence in fractions (%)(kDa) 1. NH₂-QASETGTISI a. cyclic form of glutamine → a. 23.823 no aminoacid sequence b. NH₂-QASETGTISI b. 90 b. 23.840 c. NH₂-SETGTISI c. 10 c.23.641

Example 6 Construction of Bacillus cereus 98ME1552 TruncatedMetallo-Beta-Lactamase Possessing a KT Coding Sequence Insert

In order to avoid post translational modification a molecular truncationof metallo-beta-lactamase was designed to comprise the deletion of theDNA sequence coding the NH₂-EQKLEQIVIKN region together with insertionof a KT dipeptide coding sequence located directly downstream of the3′-Hind III cloning site.

A modified metallo-beta-lactamase gene was created as in Example 5,except for the forward primer, which contained a KT coding DNA sequence.The PCR fragment was cut with Hind III and ligated to the Hind III siteof the pKTH141 secretion vector and competent B. subtilis RS303 cellswere transformed by the ligation mixture. The correct nucleotidesequence of the modified metallo-beta-lactamase gene in the expressionconstruct was confirmed by DNA sequencing. One positive clone was namedBacillus subtilis RS318 and the expression construct was called pRSH318.The nucleotide and the deduced amino acid sequence of the B. cereus98ME1552 beta-lactamase derived from pRSH318 is presented in FIG. 3, andset forth as SEQ ID NO:2 and 3, respectively.

The truncated metallo-beta-lactamase was produced, purified, andanalyzed as earlier described. The active truncated metallo enzyme waseluted from the column as a single peak. The single fraction containedmetallo-beta-lactamase that possessed a homologous amino terminalglutamic acid residue (NH₂-E; see Table 5). Accordingly, theKT-dipeptide insertion conducts post translational modificationresulting in a uniform amino terminal amino acid sequence. The truncatedmetallo-beta-lactamase was named the P2A protein.

TABLE 5 Deduced and determined amino terminal sequences and the de-termined molecular mass of truncated metallo-beta-lactamase form EnzymeDeduced NH₂- Determined NH₂- Relative proportion Mass fraction terminalamino terminal of enzyme forms in analysis n:o acid sequence sequencefractions (%) (kDa) 1. NH₂-QASKTETGTISI a. NH₂-ETGTISI a. 100 a. 23554

Example 7 Kinetic Enzyme Parameters of the P2A Metallo-Beta-Lactamase

Diversity of the catalytic properties of the P2A enzyme was studied withvarious types of beta-lactams including the penicillin family with andwithout serine beta-lactamase inhibitors, second and third generationcephalosporins, and carbapenems (meropenem). The enzyme kineticparameters k_(cat) and K_(m) were determined from initial rates by Hanesplot. The reactions were performed in 10 mM phosphate buffer, pH 7.0 at30° C. The reaction cuvette (one mL) contained about 5 pmol of enzyme inall reactions except 1.7 pmol of enzyme in the meropenem assay. Thehydrolysis of various beta-lactam substrates was spectrophometricallyrecorded at a wavelength specific for each substrate.

Values for different kinetic parameters (k_(cat), K_(m) andk_(cat)/K_(m)) that represent mean values obtained from threeindependent measurements are reported in Table 6.

TABLE 6 Kinetic parameter values for the P2A metallo-beta-lactamase TheP2A protein K_(m) k_(cat) k_(cat)/K_(m) Antibiotic (microM) (1/s) (M⁻¹ ×s⁻¹) Ampicillin 942 1114 1.18 × 10⁻⁶ Ampicillin-sulbactam 1104 1251 1.15× 10⁻⁶ (Unasyn) Amoxycillin 716 980 1.37 × 10⁻⁶ Amoxycillin-clavulanicacid 717 990 1.38 × 10⁻⁶ (Augmentin) Piperacillin 372 1049 2.82 × 10⁻⁶Piperacillin-tazobactam 412 1098 2.67 × 10⁻⁶ (Tazocin) Cefuroxime 27 2217.99 × 10⁻⁶ Cefotaxime 66 479 7.28 × 10⁻⁶ Ceftriaxone 68 95 1.40 × 10⁻⁶Meropenem 410 480 1.17 × 10⁻⁶

Example 8 Stability of the P2A Protein in Human Ileal Chyme

Resistance to intestinal proteases is one of the most important factorsaffecting the applicability of the P2A protein as a drug substance intargeted beta-lactamase therapy in the small intestine. Thesusceptibility of metallo-beta-lactamase to the action of smallintestinal proteases was tested by adding various amounts of activeenzyme in tubes consisting human ileal chyme. The hydrolysis ofmetallo-beta-lactamase was monitored by measuring beta-lactamaseactivity of ileal samples at various time points. Meropenem was employedas substrate in the activity assays.

The obtained results from four independent experiments and the meanvalues are expressed in Table 7. The P2A enzyme appears to be a stableprotein, which was cleared in human ileal chyme with a half-life of 55minutes (mean value). High variations of half-lives were observedbetween various experiments. However, the half-life of P2A in humansmall intestinal chyme has been evaluated to be adequate for successfulapplication of P2A enzyme therapy for elimination of residualbeta-lactam induced adverse reaction in the intestinal tracts.

TABLE 7 Half-life (in vitro) of the P2A metallo-beta-lactamase in humanileal chyme. Experiment n: o Half-life (minutes) Mean value (±SD) 1 6055 ± 25 2 80 55 ± 25 3 20 55 ± 25 4 60 55 ± 25

REFERENCES

-   Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z.,    Miller W., Lipman D. J. 1997. Gapped BLAST and PSI-BLAST: a new    generation of protein database search programs. Nucleic Acids Res.    25: 3389-3402-   Ambler, R. P. 1980. The structure of beta-lactamases. Philos.    Trans. R. Soc. London B 289:321-331.-   Bush, K. 1998. Metallo-β-lactamases: a class apart. Clin. Infect.    Dis. 32: 271-276.-   Carfi A, Duee E, Galleni M, Frere J M, and Dideberg O. 1998a. 1.85 A    resolution structure of the zinc (II) beta-lactamase from Bacillus    cereus. Acta Crystallogr D Biol Crystallogr. 54:313-323.-   Carfi A, Duee E, Paul-Soto R, Galleni M, Frere J M, and Dideberg O.    1998b. X-ray structure of the ZnII beta-lactamase from Bacteroides    fragilis in an orthorhombic crystal form. Acta Crystallogr D Biol    Crystallogr. 54:45-57.-   Carfi, A., Pares, S., Duee, E., Galleni, M., Duez, C., Frere, J. M.,    and Dideberg O. 1995. The 3-D structure of a zinc    metallo-beta-lactamase from Bacillus cereus reveals a new type of    protein fold. EMBO J. 14:4914-4921.-   Concha, N. O., Janson, C. A., Rowling, P., Pearson, S., Cheever, C.    A, Clarke, B. P., Lewis, C., Galleni, M., Frere, J. M., Payne, D.    J., Bateson, J. H., and Abdel-Meguld, S. S. 2000. Crystal structure    of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and    its complex with a mercaptocarboxylate inhibitor: binding    determinants of a potent, broad-spectrum inhibitor. Biochemistry.    39:4288-4298.-   Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J.,    Dideberg, O., and Frere, J. M. 2001. Metallo-beta-lactamases    Standard numbering scheme for class B beta-lactamases. Antimicrob.    Agents Chemother. 45:660-663-   Garau, G., Garcia-Saez, I., Bebrone, C., Anne, C., Mercuri, P.,    Galleni, M., Frere, J. M., and Dideberg, O. 2004. Update of the    standard numbering scheme for class B beta-lactamases. Antimicrob.    Agents Chemother. 48:2347-2349.-   Garau, G., Bebrone, C., Anne, C., Galleni, M., Frere, J. M.,    Dideberg, O. 2005. A metallo-beta-lactamase enzyme in action:    crystal structures of the monozinc carbapenemase CphA and its    complex with biapenem. J Mol Biol. 345:785-795.-   Harmoinen, J., Mentula, S., Heikkilä, M., van der Rest M.,    Rajala-Schultz, P. J., Donskey, C. J., Frias, R., Koski, P.,    Wickstrand, N., Jousimies-Somer, H., Westermarck, E.,    Lindevall, K. 2004. Oral Targeted Recombinant Beta-Lactamase    Prevents Ampicillin-Induced Selective Pressure on the Gut    Microbiota: A Novel Approach to Reduce Antimicrobial Resistance    Antimicrob. Agents and Chemotherapy. 48: 75-79.-   Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic    acid from micro-organisms. J. Mol. Biol. 3: 208-218.-   Mentula, S., Harmoinen, J., Koski, P., Westermarck, E., Huovinen,    P., and Könönen, E. 2004. Inhibition of ampicillin-induced emergence    of resistance in intestinal coliforms by targeted recombinant    beta-lactamase. Int. J. Antimicrob. Agents. 24:555-561.-   Sambrook, J., and Russell, D. W. 2001. Molecular cloning, a    Laboratory Manual. Cold Spring Harbour Laboratory Press Cold Spring    Harbour, New York.-   Stiefel, U., Pultz, N. J., Harmoinen, J., Koski, P., Lindevall, K.,    Helfand, M. S., Donskey, C. J. 2003. Oral β-lactamase administration    preserves colonization resistance of piperacillin-treated mice. J    Infect Dis. 10: 1605-1609.-   Stiefel, U., Harmoinen, J., Koski, P., Kaariainen, S., Wickstrand,    N., Lindevall, K., Pultz, N. J., Bonomo, R. A., Helfand, M. S., and    Donskey, C. J. 2005. Orally administered recombinant    metallo-beta-lactamase preserves colonization resistance of    piperacillin-tazobactam-treated mice. Antimicrob. Agents Chemother.    49: 5190-5191.

Walsh, T. R., Toleman, M. A., Poirel, L., and Nordmann, P. 2005.Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev.18:306-325

1. A modified metallo-beta-lactamase protein having the general formula:NH₂—K-T-E-ΔBL-COOH wherein K is lysine T is threonine E is glutamicacid, and ΔBL is a metallo-beta-lactamase protein that has beentruncated at the amino terminal end so as to leave four beta-strandsbefore the first alpha-helix of the predicted secondary structure ofsaid protein.
 2. An isolated nucleotide molecule comprising a nucleotidesequence encoding the metallo-beta-lactamase protein of claim
 1. 3. Thenucleotide molecule of claim 2, comprising sequential nucleotides 94-756of SEQ ID NO:5.
 4. An expression vector containing the nucleotidemolecule of claim
 2. 5. A host cell capable of expressing themetallo-beta-lactamase protein encoded by the nucleotide molecule ofclaim
 2. 6. The host cell of claim 5, which secretes saidmetallo-beta-lactamase protein.
 7. The host cell of claim 5, which isBacillus spp, especially B. licheniformis or B. subtilis.
 8. A modifiedmetallo-beta-lactamase protein having the general formulaNH₂-E-ΔBL-COOH wherein E is glutamic acid, and ΔBL is ametallo-beta-lactamase protein that has been truncated at the aminoterminal end so as to leave four beta-strands before the firstalpha-helix of the predicted secondary structure of said protein.
 9. Themetallo-beta-lactamase protein of claim 8, which belongs to subgroup B1of metallo-beta-lactamases.
 10. The metallo-beta-lactamase protein ofclaim 9, wherein the truncated beta-lactamase is derived from Bacillusspp., and especially from B. cereus.
 11. The metallo-beta-lactamase ofclaim 10, wherein E-ΔBL is derived by deleting the first eleven aminoterminal amino acids of a mature metallo-beta-lactamase.
 12. Themetallo-beta-lactamase protein of claim 9, wherein the sequence E-ΔBLhas at least 80% identity to the amino acid sequence of sequential aminoacid residues 6-221 of SEQ ID NO:3.
 13. The metallo-beta-lactamaseprotein of claim 9, wherein the sequence E-ΔBL is a truncated form of ametallo-beta-lactamase having the sequence set forth as SEQ ID NO:1, ora beta-lactamase active variant or fragment thereof.
 14. Themetallo-beta-lactamase of claim 9, wherein E-ΔBL is derived bytruncation of a metallo-beta-lactamase between the amino acids KN and E.15. A method of preparing a modified metallo-beta-lactamase protein,said method comprising culturing a host cell of claim 5 under conditionsenabling the expression of a metallo-beta-lactamase having the generalformula:NH₂—K-T-E-ΔBL-COOH, wherein K is lysine T is threonine E is glutamicacid, and ΔBL is a metallo-beta-lactamase protein that has beentruncated at the amino terminal end so as to leave four beta-strandsbefore the first alpha-helix of the predicted secondary structure ofsaid protein, and conducting post translational modification resultingin a modified metallo-beta-lactamase having the general formula:NH₂-E-ΔBL-COOH, wherein E and ΔBL are as defined above, and optionallyisolating and purifying the post translationally modified proteinobtained.
 16. The method of claim 15, wherein the metallo-beta-lactamaseprotein is expressed in a form comprising a signal sequence, whereby theprotein is secreted from the host cell.
 17. The method of claim 15,wherein the protein is produced in Bacillus spp. especially in B.licheniformis or B. subtilis.
 18. A pharmaceutical compositioncomprising the modified metallo-beta-lactamase protein of claim
 8. 19.The modified metallo-beta-lactamase of claim 8 for use as a medicament.20. Use of the modified metallo-beta-lactamase of claim 8 for themanufacture of a medicament for elimination of beta-lactam antibioticinduced adverse effects in the intestinal tract.
 21. The use of claim20, wherein the beta-lactam antibiotic is selected from the groupconsisting of cephalosporins, carbapenems and penicillins, whichantibiotic is to be eliminated in the presence or absence of aninhibitor against serine beta-lactamases.
 22. A method of treatingbeta-lactam antibiotic induced adverse effects in the intestinal tractcomprising administering an effective amount of the modifiedmetallo-beta-lactamase of claim 8 or the pharmaceutical composition ofclaim 18 to a person in need thereof.
 23. A method for the production ofa homogeneous metallo-beta-lactamase enzyme preparation comprising: a)preparing an expression construct that expresses a protein having theformula: NH₂—K-T-3-Δbl, wherein K is lysine, T is threonine, E isglutamic acid, and -ΔBL is a metallo-beta-lactamase protein that hasbeen truncated at the amino terminal end so as to leave fourbeta-strands before the first alpha helix of the predicted secondarystructure of said protein; b) transforming a bacterial host cell withsaid expression construct; and c) isolating the metallo-beta-lactamaseenzyme preparation produced by said bacterial host cell in culture,wherein said metallo-beta-lactamase enzyme preparation produced by saidbacterial host cell in culture is more homogeneous than a preparation ofnon-truncated metallo-beta-lactamase produced under similar conditionsfrom a host cell transformed with an expression construct containing anunmodified metallo-beta-lactamase encoding gene.